I. Field of the Invention
The present invention relates to a method for collecting cells in M phase (mitotic phase) or G1 phase and method for nuclear transplantation using the cells.
II. Description of the Related Art
It is important to obtain a population of cells in the same particular cell cycle for clarifying the biochemical reactions which occur in the particular cell cycle, the mechanism of cell proliferation and the like. Among the methods for synchronizing the cell cycle to metaphase of cell division, the shaking method by Terashima et al1) utilizes the fact that the cells in the metaphase of cell division are spherical and their adhesiveness with the culture plate is small, so that they are easily peeled off therefrom, which enables collection of the cells synchronized to metaphase of cell division without changing the physiological state. In their method, a portion of the culture medium is lightly blown to the surface of the culture medium with a pipette, and the cells detached thereby are collected. Among the thus collected cells, 76.6 to 87.4% thereof are in the M phase of cell cycle, but a population of cells which are entirely in the metaphase cannot be collected by their method.
On the other hand, for synchronizing the cell cycle to metaphase, colchicin2),3), colcemid4), nocodazole5) or the like is usually employed. Zieve et al6) treated the cells with nocodazole and collected cells by the shaking method to obtain cells in the metaphase in an amount of 25 to 34%. However, the efficiency of collecting the cells in metaphase is thus low.
By the recent nuclear transplantation technology using somatic cells, a viable offspring was successfully produced using the nuclei of the cells induced to G0/G1 phase by serum starvation. This technology is also useful for investigating behavior of the nuclei of the embryos whose nuclei were transplanted from cells in a particular cell cycle, and for clarifying the mechanism of development. That is, in 1997, Wilmut et al succeeded in producing a viable offspring by nuclear transplantation using somatic cells7). In their method, the cell cycles of the cells to be used as nuclear donors are synchronized to G0/G1 phase by culturing sheep mammary gland cells, and by decreasing the serum concentration in the culture medium from 5% to 0.05% after growing the cells, thereby subjecting the cells to serum starvation. This method is now widely used in preparation of nuclear donors. However, this method has a drawback in that fragmentation of DNAs in the cells subjected to the serum deprivation more frequently occurs than the cells not subjected to the serum deprivation8),9), so that the percentage of the surrogate mothers who cannot continue pregnancy is high10) when the cells subjected to serum deprivation are used as the nuclear donors. As a result, the efficiency of producing somatic clone is low, which is problematic.